: Recent advances in our understanding of the molecular and genetic basis of primary immune deficiency syndromes have made these disorders prime candidates for gene therapy. In the majority of the genetically determined immune deficiency syndromes, the gene has been identified, mutations causing the syndromes have been found, and the molecular consequences on the immune response recognized. The overall goal of this project is to develop techniques to correct the genetic defect in X-linked hyper IgM syndrome (XHIM), caused by the mutation of the CD40 ligand (CD40L) gene, and to work out the principles for gene therapy of their primary immunodeficiency disorders. Currently, retroviral vectors offer the most attractive method for the transfer of genes into target cells that can easily be cultured, transduced and, potentially, returned to the donor, as outlined in this proposal. We have constructed the retroviral vector, LCD40LSN in which the human CD40L gene is expressed from the viral LTR, and transduced the gene with high efficiency into normal human fibroblasts that untreated do not express CD40L. In Aim 1, we propose to produce a series of retroviral vectors that will result in either constitutive expression or activation-induced expression of CD40L. In addition to the LTR-containing vector, we will use the weaker SV40 promoter for constitutive expression, and the human IL-2 and CD40L promoters for activation-induced CD40L expression. In Aim 2, we will determine the conditions for high efficiency transduction of T lymphoblastoid cell lines derived from XHIM patients who have mutations that either interfere with gene expression or result in the expression of a nonfunctional protein. The transduced T cell lines will be assessed for CD40L expression by flow cytometry and for CD40L function by an in vitro tonsillar B cell culture system. In Aim 3, we will use the most suitable vectors identified by in vitro experiments to transfer murine CD40L gene in bone marrow stem cells of CD40 ligand knockout mice. This will allow us to test the in vivo effect of consituitively expressed CD40L vis-a-vis activation-induced expression of CD40L on the immune system and on the clinical phenotype of recipient mice. The studies will pave the way for gene therapy in patients will genetically determined primary immunodeficiency diseases.